1. Field of the Invention
A process for making DNA libraries in filamentous fungal cells using a novel cloned gene involved in the mismatch repair system of filamentous fungal cells.
2. Description of the Related Art
The mismatch repair system is a system within cells which recognizes mismatches in newly synthesized duplex DNA sequences.
The mismatch repair system then either corrects the mismatches which are seen as errors by e.g. using the methylated xe2x80x9coldxe2x80x9d strain as template or alternatively it may mediate degradation of the duplex DNA sequences which comprise the mismatches.
Independently on the precise mechanism the end result will be that the xe2x80x9cmismatch repair systemxe2x80x9d will limit the xe2x80x9cdiversityxe2x80x9d within a cell, diversity being represented as duplex DNA sequences which comprise mismatches.
For example a duplex DNA sequence which comprises a single mismatch represents a diversity of two different DNA sequences within the cell. If the mismatch repair system corrects the mismatch there will only be a diversity of one within the cell.
Alternatively, if the mismatch repair system mediates the degradation of such a duplex DNA sequence the diversity will be lost. See FIG. 1 for a graphic illustration on how the mismatch repair system may work within a cell.
Consequently, if duplex DNA sequences comprising mismatches represent a DNA library of interest, then the diversity of this library may be limited when transformed (placed) into cells with an active mismatch repair system.
The art provides a solution to this problem by making cells wherein the mismatch system is inactive.
EP 449923 describes bacterial cells wherein the mismatch system is inactivated.
WO 97/37011 describes yeast cells wherein the mismatch system is inactivated. See the working examples of this document.
WO 97/05268 describes mice cells wherein the mismatch system is inactivated. See the working examples of this document.
The problem to be solved by the present invention is to provide an improved strategy for making DNA libraries in filamentous fungal cells. A filamentous fungal cell population comprising such a DNA library may then be used to select a polypeptide of interest. Also polynucleotide sequences with particular properties might be selected, such as promoters, terminators and other regulatory elements with changed/improved properties.
The solution is based on that the present inventors have cloned a novel gene involved in the mismatch repair system of a filamentous fungal cell. Further, this gene is the first gene cloned which is involved in the mismatch repair system of a filamentous fungal cell.
By inactivating this gene in a filamentous cell it is possible to obtain a filamentous cell which is deficient in its mismatch repair system and which is highly useful for preparing DNA libraries in filamentous fungal cells.
The gene comprises a very characterizing DNA sequence encoding the polypeptide sequence shown in positions 683-758 of SEQ ID NO:2.
This DNA has been used to clone the full length gene encoding the polypeptide sequence shown in positions 1-940 of SEQ ID NO:2. See working examples herein (vide infra).
The gene cloned as described in working examples herein is a gene cloned from an Aspergillus oryzae filamentous fungal cell.
However, based on the novel sequence information provided herein it is routine work for the skilled person to clone similar homologous genes from other filamentous fungal cells by, e.g., standard hybridization or PCR technology, preferably by using the DNA sequence encoding the polypeptide sequence shown in positions 683-758 of SEQ ID NO:2 as a basis for making a hybridization probe or PCR primers.
Accordingly, in a first aspect the present invention relates to a filamentous fungal cell, wherein a gene involved in the mismatch repair system has been inactivated and in which the gene involved in the mismatch repair system comprises:
(a) a DNA sequence encoding the polypeptide sequence shown in positions 683-758 of SEQ ID NO:2; or
(b) a DNA sequence encoding a polypeptide sequence which is at least 70% identical to the polypeptide sequence shown in positions 683-758 of SEQ ID NO:2; and
in a second aspect the present invention relates to a filamentous fungal cell, wherein a gene involved in the mismatch repair system has been inactivated and in which the gene involved in the mismatch repair system comprises:
(a) a DNA sequence encoding the polypeptide sequence shown in positions 1-940 of SEQ ID NO:2; or
(b) a DNA sequence encoding a polypeptide sequence which is at least 70% identical to the polypeptide sequence shown in positions 1-940 of SEQ ID NO:2.
As stated above a filamentous fungal cell of the first or second aspect of the invention is very suitable for making a DNA library of interest in filamentous fungal cells.
Accordingly, in a third aspect the present invention relates to a process for preparing a filamentous fungal cell population wherein individual cells in the population comprise individually different DNA sequences of interest representing a DNA library of interest comprising the following steps:
(a) placing individually different DNA sequences of interest in a filamentous fungal cell population comprising a filamentous fungal cell of the first or second aspect of the invention; and
(b) growing the population of (a) for a period of time allowing an individual DNA sequence of interest in the population to be duplicated at least once under conditions wherein the mismatch repair system gene of the first or second aspect of the invention has been inactivated.
One of the advantages of allowing an individual mismatch repair inactivated filamentous fungal cell duplicated DNA of interest at least once as described under step (b) of the third aspect is illustrated in FIG. 1. As can be seen in FIG. 1 the process of the third aspect using a filamentous fungal mismatch repair inactivated cell as described herein allows preparation of a DNA library wherein eventual hetero duplex DNA mismatches are not corrected. This gives a DNA library with a higher diversity as compared to a DNA library made in a filamentous fungal cell NOT having an inactivated mismatch repair system (see FIG. 1). Duplication of DNA sequence of interest means that the two strands are replicated such that two separate sets of double stranded DNA are generated, each being based on a separate one of the two original strands.
A filamentous fungal cell population wherein individual cells in the population comprise a DNA library of interest as described above may be used to select a polypeptide of interest.
Accordingly, in a fourth aspect the present invention relates to a process for production of a polypeptide of interest comprising the steps of the third aspect and wherein the DNA sequences of interest encode a polypeptide of interest and which further comprises the following step:
(c) selecting from the resultant population of filamentous fungal cells of step (b) of the third aspect a desired polypeptide of interest.
An advantage of the process of the fourth aspect may be that the polypeptide of interest is selected from a filamentous fungal cell expressing the polypeptide. Consequently, it is directly known that the polypeptide can be expressed from a filamentous fungal cell, which is useful if it is subsequently required to produce the polypeptide in large scale in a filamentous fungal cell. This may be of particular interest when the DNA library encodes polypeptides of interest which are derived from filamentous fungal cells, since it is known that filamentous fungal polypeptides preferably are produced in industrial relevant high yields in filamentous fungal cells.
This is contrary to a similar selection process using e.g. a yeast cell. Here the only thing known is that the selected polypeptide is capable of being expressed in yeast and later expression a filamentous fungal cell might give problems, especially if high yields are required.
Definitions
The following section provides definitions of technical features in above-mentioned aspects of the invention.
The term xe2x80x9ca genexe2x80x9d denotes herein a gene (a DNA sequence) which is capable of being expressed into a polypeptide within the cell. Accordingly, the gene sequence will be defined as an open reading frame starting from a start codon (normally xe2x80x9cATGxe2x80x9d, xe2x80x9cGTGxe2x80x9d, or xe2x80x9cTTGxe2x80x9d) and ending at a stop codon (normally xe2x80x9cTAAxe2x80x9d, TAGxe2x80x9d or xe2x80x9cTGAxe2x80x9d).
In order to express the gene there must be elements, as known in the art, in connection with the gene, necessary for expression of the gene within the cell. Such standard elements may include a promoter, a ribosomal binding site, a termination sequence, and may be other elements as known in the art.
The term xe2x80x9cmismatch repair systemxe2x80x9d shall herein be understood according to the art, as a system within cells which recognizes mismatches in duplex DNA sequences. (See e.g. WO 97/37011, page 1, line 21-28)
The mismatch repair system then either corrects the mismatches which are seen as errors by e.g. using the methylated xe2x80x9coldxe2x80x9d strain as template or alternatively it may mediate degradation of the duplex DNA sequences which comprise the mismatches.
Independently on the precise mechanism the end result will be that the xe2x80x9cmismatch repair systemxe2x80x9d, will limit the xe2x80x9cdiversityxe2x80x9d within the cell represented by such duplex DNA sequences which comprise mismatches.
For example, a duplex DNA sequence which comprises a single mismatch represents a diversity of two different DNA sequences within the cell. If the mismatch repair system corrects the mismatch their will only be a diversity of one within the cell. Alternatively, if the mismatch repair system mediates the degradation of such a duplex DNA sequence this diversity will be lost.
A polypeptide encoded by a gene involved in the mismatch repair system recognizes a mismatch by a mechanism involving binding to the mismatch.
Accordingly, a suitable assay to test whether or not a filamentous fungal cell as described herein is inactivated in its mismatch repair system is to use a xe2x80x9cgel shift assayxe2x80x9d or alternatively termed a xe2x80x9cgel retardation assay.xe2x80x9d This is a standard assay used in the art. See WO 97/05268, pages 16, 17 and 25.
The principle in such an assay is that cell extracts are prepared of both (a) a filamentous fungal cell wherein the gene, as described herein, involved in the mismatch repair system is inactivated; and (b) the corresponding filamentous fungal cell wherein the gene is NOT inactivated. These extracts are then bound/mixed to oligonucleotides containing the base-pair mismatched G:T; G:A; G:G; A:C, and an extrahelical TG dinucleotide and run on a nondenaturing gel.
If the gel shift assay demonstrates that the control filamentous fungal cell wherein the gene is NOT inactivated comprises any protein(s) which binds to any of above mentioned oligonucleotides and these binding protein(s) are NOT seen in the filamentous fungal cell wherein the gene, as described herein, involved in the mismatch repair system is inactivated then it is a confirmation that the mismatch repair system in the latter is inactivated.
A detailed description of a suitable gel shift assay is provided in working example 1 herein.
The sequence identity in relation to the phrases
xe2x80x9ca DNA sequence encoding a polypeptide sequence which is at least 70% identical to the polypeptide sequence shown in positions 683-758 of SEQ ID NO:2xe2x80x9d and
xe2x80x9ca DNA sequence encoding a polypeptide sequence which is at least 70% identical to the polypeptide sequence shown in positions 1-940 of SEQ ID NO:2xe2x80x9d;
is determined as the degree of identity between two sequences indicating a derivation of the first sequence from the second. The identity may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-453). Using GAP with the following settings for polypeptide sequence comparison: GAP creation penalty of 3.0 and GAP extension penalty of 0.1, the polypeptide encoded by an analogous DNA sequence of the invention exhibits a degree of identity preferably of at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and especially at least 97% with amino acid sequence shown in positions 683-758 of SEQ ID NO:2, according to the first aspect of the invention; or with amino acid sequence shown in positions 1-940 of SEQ ID NO:2, according to the second aspect of the invention.
The term xe2x80x9cDNA libraryxe2x80x9d denotes herein a library of at least two different DNA sequences. For many practical purposes the library is much bigger. Accordingly, the DNA library preferably comprises at least 1000 different DNA sequences, more preferably at least 10000 different DNA sequences, and even more preferably at least 100000 different DNA sequences.
The term xe2x80x9cplacing individually different DNA sequences of interest in a filamentous fungal cell populationxe2x80x9d in relation to step (a) in the process of the third aspect of the invention shall herein be understood broadly in the sense that it is NOT identical DNA sequences of interest which are placed in the filamentous fungal cell population. In the present context, relating to a process for making a DNA library using a mismatch repair deficient cell, the term should preferably denote a situation wherein a cell within the filamentous fungal cell population comprises at least two different DNA sequences of interest which are so partially homologous that they are capable of hybridizing/recombining to each other within the cell. It is within the skilled person""s general knowledge to determine how partially homologous such sequences have to be in order to obtain said recombination within the cell.
A practical example may be that single stranded oligonucleotide sequences partially homologous to chromosomal DNA sequence are placed within the cell or duplex DNA sequences comprising mismatches (e.g. comprised within a vector) are placed within the cell. See below for further description of such examples.
The specific experimental way of placing these DNA sequences within a filamentous cell may be done according to any of the many suitable techniques, such as transformation techniques.
The phrase xe2x80x9cgrowing the population of (a) for a period of time allowing an individual DNA sequence of interest in the population to be duplicated at least once under conditions wherein the mismatch repair system gene has been inactivatedxe2x80x9d according to step (b) of the third aspect of the invention denotes that after an individual cell has duplicated itself at least once then the mismatch repair system may be activated again without losing the advantage of the process. The technical reason for this is illustrated in FIG. 1. In this example a duplex DNA sequence comprising a single mismatch is placed in a filamentous cell. After the cell has been duplicated once under conditions wherein the mismatch repair system gene has been inactivated the two individually different single stranded DNA sequences within the duplex DNA have individually been duplicated providing two different duplex sequences, one in each duplicated cell, without any mismatches. Accordingly, since such a cell does NOT comprise duplex DNA sequences of interest having mismatches then there is no technical need for maintaining the mismatch repair system inactivated.
In sections below are described preferred embodiments of the invention by way of examples only.